A fully validated isotope dilution HPLC‐MS/MS method for the simultaneous determination of succinylcholine and succinylmonocholine in serum and urine samples

Abstract

A high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous detection of succinylcholine (SUX) and its metabolite succinylmonocholine (SMC) in serum and urine is presented. For internal standardization using isotope dilution, the deuterated compounds SUX-d(18) and SMC-d(3) were employed. Full validation was performed according to international guidelines. Solid-phase extraction (SPE) of acidified samples was accomplished using Strata-X polymeric reversed phase cartridges together with heptafluorobutyric acid (HFBA) as ion-pairing reagent. Separation was achieved within 13 min on a Phenomenex Synergi Hydro RP C18 column (4 microm, 150 x 2 mm) using a gradient of 5 mM ammonium formate buffer pH 3.5 and acetonitrile.To ensure the method's applicability in forensic as well as clinical toxicology, the specific demands of both research fields were taken into account, and the method was thus validated for a low and high concentration range. For both serum and urine as sample matrix, the validation revealed good intraday and interday precisions, consistently ranging below 15% for the lowest and below 10% for elevated concentrations. Accuracy was likewise good and never exceeded 10%. Extraction recovery was excellent, ranging between 88.1 and 103.9% for SUX and SMC in both tested matrices. Matrix effects were significant, the otherwise optimized extraction and detection methods, however, allowed for a very satisfactory sensitivity of the described method: For serum, the limits of detection and quantitation were determined to be 1.9 and 6.0 ng/ml for SUX, as well as 2.5 and 8.6 ng/ml for SMC, respectively; for urine, the corresponding values were established to be 1.4 and 4.0 ng/ml (SUX), as well as 1.5 and 4.9 ng/ml (SMC).The presented method was successfully applied to authentic samples of two forensic cases investigated in the institute of forensic medicine in Bonn, allowing the diagnosis of SUX intoxications.

Succinylmonocholine analytics as an example for selectivity problems in high‐performance liquid chromatography/tandem mass spectrometry, and resulting implications for analytical toxicology

“…12 Succinylcholine, paraoxon, heptafluorobutyric acid (HFBA) and ammonium formate were obtained through Sigma Aldrich (Schnelldorf, Germany). Methanol as well as acetonitrile were of HPLC grade and obtained from varying sources.…”

“…With a chromatographic approach failing to yield acceptable results, Allen alternatively proposes introduction of an MRM transition for O-desmethyltramadol, to ascertain tramadol presence via detection of its metabolite. Our group recently developed an HPLC/electrospray ionization (ESI)-MS/MS method for the parallel detection and quantitation of succinylcholine (SUX) and succinylmonocholine (SMC), 12 during the development of which we were confronted with similar selectivity concerns. By presenting initial findings, further investigation into the subject as well as the final solution of the problem, the present work aims at calling attention to the yet underrepresented selectivity problems in HPLC/MS/MS, and highlights the absolute necessity to monitor more than just one ion transition for a reliable analyte detection.…”

Pharmacokinetic Properties of Succinylmonocholine in Surgical Patients

“…Sample aliquots (5 µL) were injected for analysis, and gradient elution using mobile The herein described analytical method has been fully validated as published previously (11). Limits of detection and quantitation were well below 10 ng/mL for both target substances; precision and accuracy were constantly below 15% for the whole concentration range (12.5 ng/mL-100 µg/mL) (11).…”

“…Analysis of samples was carried out according to a previously published method for the parallel detection of SUX and SMC (11). Briefly, SPE was performed using the Strata-X (33 µm, 200 mg/3 mL) polymeric reversed-phase SPE cartridges from Phenomenex.…”

“…These compounds were subsequently used in the validation of a sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-MS-MS method following an effective, yet fast and easy, ion-pairing solid-phase extraction (SPE) procedure (11). Having previously established that no endogenous SMC is present in serum or urine (12), the present work employs these analytical tools to monitor the in vivo degradation process of SMC, aiming to define significant detection windows for this metabolite in plasma, and establish a useful kinetic model for forensic toxicological casework.…”